PROJECT SUMMARY Preimplantation genetic testing for aneuploidy screening (PGT-A) is a major advancement in IVF technology as it provides a method for selecting against aneuploid embryos prior to transfer. The current PGT-A technology uses next generation sequencing (NGS) and can determine the percentage of aneuploid cells present in a trophectoderm (TE) biopsy. The NGS approach has confirmed that 40-60% of IVF derived blastocysts are aneuploid with most aneuploid blastocysts being composed of euploid and aneuploid cells (i.e. mosaic embryos). Mosaic blastocysts with between 20 and 40% aneuploid cells are now offered for transfer in some clinics, if euploid blastocysts are not available. Somewhat surprisingly, a relatively high percentage (? 40% of 143 embryos) of mosaic blastocysts have resulted in live births. These observations suggest that the preimplantation embryo selectively deletes aneuploid cells of the inner cell mass (ICM), which gives rise to the fetus. That the selective depletion of aneuploid cells from the ICM occurs was demonstrated using mouse mosaic blastocysts. These studies demonstrated that the number of aneuploid cells in the ICM but not in the TE decreases during blastocyst expansion. The mechanism by which the aneuploid cells are selectively deleted from the ICM is not known. We hypothesize that this mechanism involves the following features: 1) aneuploid cells in both the TE and ICM are predisposed to undergo p53-dependent apoptosis, as is the case for somatic aneuploid cells, 2) cells within the TE but not the ICM express lactate (MCT1) and sodium- hydrogen ion (NHE1) transporters. Their expression enable both the euploid and aneuploid cells of the TE to maintain a neutral intracellular pH during blastocyst expansion while metabolizing glucose through the glycolytic pathway, 3) glycolytic metabolism increases lactate and hydrogen ion content of the blastocoel fluid and causes a slight decrease in intracellular pH of ICM cells and 4) the decrease in intracellular pH activates the preexisting p53 signaling pathway, which leads to a demise of the aneuploid cells of the ICM. Experiments in this grant proposal will use mosaic mouse blastocysts to test our novel hypothesis and are organized into three specific aims. Specific aim 1 will determine whether aneuploid cells undergo apoptosis through a p53- dependent mechanism during blastocyst expansion by monitoring the expression of the components of the p53 pathway as well as generating p53 depleted aneuploid cells. Specific aim 2 will determine whether aneuploid cells that reside in TE are more resistant to apoptosis due to their ability to adapt to metabolic changes because they express MCT1 and NHE1. The role for these transporters will be demonstrated by depleting MCT1 and NHE1 in the aneuploid cells. Specific aim 3 will determine whether supplementing the culture media with weak acids or glucose and lactate will decrease the intracellular pH and selectively induce apoptosis of aneuploid cells of the ICM. The effectiveness of these treatments will be monitor by determining changes in the number of aneuploid cells in the TE and ICM and their intracellular pH.